Numerous compounds showing antineoplastic activity have been identified in the organic solvent extracts of plant material, microorganisms and marine organisms. Further investigation into the utility of these compounds as chemotherapeutic and anti-HIV agents has been hampered by their low natural natural abundance and the inefficiencies of conventional extraction techniques. An example of one such compound is taxol (NSC 125973), a diterpene plant product derived from the western yew Taxus brevifolia. This drug, currently in clinical trials, has exhibited a striking 30 to 40% response rate against advanced cases of ovarian and a number of other cancers. This drug is, however, in short supply.
Currently, taxol is extracted with organic solvents from the milled bark of T. brevifolia in three major steps and chromatographically purified in four major steps. The extraction process begins with milled bark which is percolated three times with methanol at 50.degree. to 55.degree. C. The extract is then concentrated in methanol. Next, the concentrated methanol extract is partitioned between methylene chloride and water. The methylene chloride fraction, containing taxol, is concentrated. The methylene chloride concentrate is dissolved in 50/50 acetone:hexane, and the mixture is filtered to remove insolubles. This organic extraction process yields an amber colored syrup with a solids content which is about 1.3% of the milled bark.
The conventional purification is performed in four major steps. First, the acetone:hexane mixture from the extraction process is chromatographed on Florisil columns in a 70/30 hexane:acetone mixture to separate the taxol containing fractions. The taxol fractions are then concentrated to dryness. This step may be repeated as many as nine times. Second, taxol concentrates are crystallized from a methanol:water mixture and then recrystallized from an acetone:hexane mixture yielding 85 to 95% pure taxol. Third, the taxol is chromatographed on silica gel packed with either 2.5% isopropanol or 2.5% n-butanol in methylene chloride to yield approximately 98% pure taxol. Fourth, the taxol is dissolved in acetone, the solution filtered, and taxol recrystallized from an acetone:hexane mixture.
This organic phase extraction and chromatographic purification process yields 99% pure taxol which is about 0.014% of the milled bark. The production of taxol by this technique is encumbered by the following: (i) time consuming extraction and purification procedures; (ii) long residence times in a harsh environment; and (iii) low overall yields. Also the bark of T. brevifolia is usually obtained from mature trees (100 to 200 years old). The bark is difficult and costly to harvest, and is in limited supply. That tree is in danger of extinction. A significant amount of research is being conducted to find alternate manufacturing routes and raw material sources. Some of the alternate manufacturing routes include total synthesis and semi-synthesis from a closely-related taxoid, 10-deacetyl baccatin III. Alternate sources include needles of the ornamental yew, twigs, sprouts and plant cell cultures. With the exception of total synthesis which has not yet been accomplished and which may prove difficult to commercialize, these techniques all require extraction and purification of taxoids from a biomass source.
The needle of the ornamental yew may prove to be a very attractive raw material resource. The needle contains high quantities of taxoids, and is a renewable resource which can be readily cultivated in nurseries and farms.
However, the needles present additional processing challenges. The needles contain a significant amount of waxes and nonpolar constituents which are extracted into organic solvents. Some of the more nonpolar waxes can be removed by a solvent prewash with hexane. However, some compositions closely related to taxoids coextract with taxol in the major conventional solvent extraction step using methylene chloride and methanol mixtures. The total mass extracted from the needles by organic solvent may range from 35 to 42% compared to 25% for the bark. The vastly larger percentage is not an indication of greater amount of taxol but of the impurities which coextract. The virgin needles also contain a substantial amount of water (about 60%).
The taxol extraction and purification steps for needles thus become much more complicated and time consuming than the previously described process for bark.